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目的:探讨多重连接探针扩增技术(MLPA)在无精子症及严重少精子症不育男性无精子因子(AZF)微缺失筛查中的应用可能。方法:提取147例无精子症或严重少精子症患者及154例正常对照男性外周血DNA,经95℃变性后与设计合成的AZF区域探针特异杂交,杂交产物经连接酶连接后用带有FAM荧光标记的通用引物扩增,毛细管电泳将产物分离生成MLPA图谱。所有样本同时行AZF序列标签位点(STS)的多重PCR分析。结果:病例组STS缺失检出率为15.0%(22/147),对照组中未检出STS缺失者;MLPA法于病例组中检出40例AZF区探针缺失患者,检出率为27.2%,其中包括22例STS缺失型患者。对照组中亦有20例AZF探针缺失者。结论:相比较传统的多重PCR,MLPA技术在AZF微缺失筛查中具有更佳的检测灵敏度,同时MLPA图谱所展现的高分辨的AZF区遗传学信息将有助于男性生精障碍病因学机制的深入探索。
Objective: To explore the possibility of application of multiplex ligation probe amplification (MLPA) in the screening of azoospermia (AZF) microdeletion in azoospermia and severe oligospermia. Methods: DNA from 147 cases of azoospermia or severe oligospermia and 154 normal controls were collected. After denaturation at 95 ℃, the specific probes were designed and synthesized. The hybridization products were ligated by ligase, FAM fluorescence-labeled universal primer amplification, capillary electrophoresis product separation MLPA generated. Multiplex PCR analysis of all AZF sequence tag sites (STS) simultaneously. Results: The detection rate of STS deletion was 15.0% (22/147) in the case group and no STS deletion was found in the control group. 40 patients with AZF deletion were detected by MLPA in the case group, the detection rate was 27.2 %, Including 22 cases of STS-deficient patients. There were also 20 cases of AZF probe deletion in the control group. CONCLUSION: Compared with traditional multiplex PCR, MLPA has better detection sensitivity in AZF microdeletion screening, and genetic information of high-resolution AZF region demonstrated by MLPA will be helpful to etiology of male spermatogenesis In-depth exploration.